Characterization of genes encoding diterpene cyclases responsible for phytoalexin biosynthesis in rice.

从水稻中分离到14个二萜类植物抗毒素；Momilactones， phytocassanes和oryzalexins。我们已经分离出编码二萜环化酶的cdna，这些环化酶负责这些植物抗毒素的生物合成；OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8, OsKSL10。本项目为环化酶基因的鉴定。1)基因表达模式：OsCPS2、OsCPS4、OsKSL4、OsKSL7和OsKSL8在根中的表达量高于茎中的表达量。从抽穗期到开花期，水稻果实中OsCPS2、OsCPS4、OsKSL4和OsKSL7的表达量急剧增加。绿色荧光蛋白瞬态分析表明，这些二萜环化酶是转运的薄质体。2)同源物的分离：从水稻KS类同源cdna中分离到分别编码t-皮玛拉-8,15-二烯合成酶和t-kaur-15-烯合成酶的OsKSL5和OsKSL6。从小麦等禾科植物中分离到2个同源的OsCPS2和OsCPS4 cdna。3)水稻二萜抗菌素与致病耐受性的关系：6个基因在稻瘟病病菌侵染后表达水平升高。我们没有成功筛选tos17插入敲除突变体和通过RNAi产生敲除突变体。4) OsCPS2与负责赤霉素生物合成的OsCPS1的比较：OsCPS2的异位表达挽救了缺乏OsCPS1的严重矮化表型突变体。这一结果表明，OsCPS1的表达调控与OsCPS2的表达调控不同。OsCPS2的基本酶学性质与OsCPS1基本相同，如二价阳离子需求、最适pH、最适温度和动力学参数。然而，OsCPS2的活性受到底物香叶基二磷酸香叶基和赤霉素生物合成抑制剂AMO 1618的抑制作用小于OsCPS1。

From rice 14 diterpenoid phytoalexins were isolated; momilactones, phytocassanes, and oryzalexins. We have isolated cDNAs encoding diterpene cyclases responsible for biosynthesis of these phytoalexins; OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8, OsKSL10. This project was for characterization of the cyclase genes. 1) Gene expression patterns: The expression levels in roots of OsCPS2, OsCPS4, OsKSL4 OsKSL7, and OsKSL8, were higher than those in shoots. The expression levels of OsCPS2, OsCPS4, OsKSL4, and OsKSL7 were drastically increased in the rice fruits from heading stage to flowering stage. Transient assay using green fluorescence protein suggested that these diterpene cyclases are transported thin plastids. 2) Isolation of their homologues: From rice KS like homologue cDNAs, OsKSL5 and OsKSL6, which encode ent-pimara-8,15-diene synthase and ent-kaur-15-ene synthase respectively, were isolated. Two homologue cDNAs of OsCPS2 and OsCPS4 were isolated from wheat, other species in Poaceae. 3) Relationship of rice diterpene phytoalexins with pathogenic tolerance: The expression levels of six genes increased after infection by Magnaporthe grisea. We did not succeed in screening Tos 17-insertion-knock-out mutants and production of knock-down mutants by RNAi. 4) Comparison of OsCPS2 to OsCPS1 that is responsible for gibberellin biosynthesis: The severe dwarf phenotype of mutants lacking in OsCPS1 were rescued by ectopic expression of OsCPS2. This result indicated that regulation of OsCPS1 expression is different from that of OsCPS2 expression. The basic enzymatic properties of OsCPS2 were almost same as those of OsCPS1, such as divalent cation requirement, optimal pH, optimal temperature and kinetic parameter. However, the activity of OsCPS2 was suppressed by both of substrate geranylgeranyl diphosphate and a gibberellin-biosynthesis-inhibitor AMO 1618 less than that of OsCPS1.